There are numerous methods for chemically changing and/or fixing the collagenous matrix of biological tissues to enable such tissues to be implanted into a living mammalian body. Examples of changed and fixed implantable biological tissues include cardiac valves, blood vessels, pericardium, skin, dura mater, tendons and ligaments.
These biological tissues consist mainly of collagen and elastin. The rigidity/elasticity of most biological tissues is largely determined by the relative collagen and elastin content in the tissues and/or the physical configuration of the connective tissue framework.
Each collagen molecule consists of three polypeptide chains intertwined to form a coiled triple helix. Chemical agents used to preserve biological tissues generally form cross-links between amino groups situated on the polypeptide chains within a collagen molecule (intramolecular) as well as between adjacent collagen molecules (intermolecular).
Collagen-based biomaterials, when used as implantable devices in different recipient species, are prone to hyperacute rejection. This hyperacute rejection is a natural immunological response, triggered by antigens present in the structure of the collagen-based biomaterial. Hyperacute rejection is a rapid degenerative process, which affects the function and durability of such an implantable device.
The antigenicity of collagen-based biomaterials can be suppressed by physical or chemical cross-linking of the collagen. Physical cross-linking methods such as ultraviolet irradiation or thermal dehydration results in low density cross-linking. Chemical agents such as formaldehyde, glutaraldehyde, dialdehyde starch and certain polyepoxy compounds have been used as chemical cross-linking agents in collagen-based biomaterials.
Cross-linking collagen involves the reaction of a cross-linking agent with amine groups of lysine or hydroxylysine residues on different polypeptide chains. Another known method of cross-linking collagen is to activate the carboxyl groups of glutamic and aspartic acid residues in a polypeptide chain to react with the amine groups of another polypeptide chain to form amide bonds.
Cross-linking can also be performed by bridging amine groups of adjacent polypeptide chains with diisocyanates, which results in the formation of urea bonds. This method is less popular due to the toxicity and the low solubility of most diisocyanates.
In recent times, glutaraldehyde has been the cross-linking agent of choice. Glutaraldehyde is rendered bifunctional due to the presence of an aldehyde present at both ends of a five carbon aliphatic chain. Apart from fixing the tissue, glutaraldehyde is an excellent sterilising agent for preparing biological tissues for implantation.
In particular, permanently implantable biomaterials, which have been fixed with glutaraldehyde, include porcine bioprosthetic heart valves, bovine pericardial valves and bovine pericardial patches.
A problem associated with the implantation of biological materials, cross-linked with chemical agents, is that these materials, specifically the collagen and elastin in these materials, tend to calcify. Calcification of these materials can result in stiffening which result in degradation and failure of the material. It is known that both extrinsic and intrinsic calcification is responsible for the calcification of cross-linked biomaterials.
Unfortunately, glutaraldehyde is known to promote calcification in biomaterials. Reaction of aldehyde and primary amines in the biomaterials form unstable imines (Schiff base) which subsequently release glutaraldehyde from the biomaterial. Unbound aldehydes present in the tissue can cause severe tissue irritations, such as inflammatory reactions, after implantation. There is therefore a need to remove or inactivate the calcification-promoting effects of cross-linking agents such as glutaraldehyde.
The mechanism of calcification of cross-linked biomaterials has not yet been fully understood. Clinical data have shown that factors such as patient age, infection, host tissue chemistry, dehydration, distortion, dietary factors and inadequate initial anticoagulation therapy can promote calcification of implanted biomaterials.
Many attempts have been undertaken to find ways to mitigate the calcification of cross-linked biomaterials. Research on the mitigation of calcification of biomaterials has primarily focussed on the treatment of the cross-linked biomaterials and is described in, but not limited to, U.S. Pat. No. 4,553,974 (Dewanjee et al.); U.S. Pat. No. 4,120,649 (Schechter); U.S. Pat. No. 4,648,881 (Nashef et al.); and U.S. Pat. No. 4,976,733 (Girardot) Vyavahare et al., 1997, Circulation, 95:479-488 and Pathak et al., 2004, J. Biomed. Mater Res., 69A: 140-144. These publications generally describe methods of treating fixed tissues with alcohol before implantation. In other words, the tissue has already been cross-linked before being exposed to alcohol. Even in instances where tissues are pre-incubated in the presence of alcohol, the period of exposure is often too short to be useful or the presence of buffer and other agents adversely affects the cross-linking stability (see, for example, Vyavahare et al., 1997, supra). Alternative processes to fix biomaterials with non-glutaraldehyde reagents have also been described and these include, but are not limited to, the use of polyglycidal ethers (Imamura et al., (1988), Jpn. J. Artif. Organs, 17:1101-1103); photo-oxidation (Moore et al., (1994), J. Biomed. Mater. Res., 28:611-618).
Treatment of cross-linked biomaterials with amino-di-phosphate and surfactant has demonstrated reduced calcification in these biomaterials after implantation. However, these agents tend to wash out of the biomaterial after implantation and only delay the calcification process.
The use of alcohol in the treatment of biomaterials is well known, but is limited to its use as a solvent and/or sterilising agent. For example, the use of alcohol in the treatment of biomaterials against pathologic calcification is limited to its use in previously cross-linked collagenous biomaterials; U.S. Pat. No. 5,746,775 (Levy et al.) and International Pat. No. WO84/01894.
Consequently, there still exists a need for a method of producing a biomaterial that has a long-term resistance to calcification.